RAE1 Ligands for the NKG2D Receptor Are Regulated by STING-Dependent DNA Sensor Pathways in Lymphoma

Abstract

The immunoreceptor NKG2D originally identified in natural killer cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DLs) is activated by the DNA damage response (DDR) which is often activated constitutively in cancer cells, revealing them to natural killer cells as a mechanism of immunosurveillance. Here we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines which express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3+/−;Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared to Irf3+/+;Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance.

Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

“…Increasing the intensity of the PicoGreen signal revealed weaker PicoGreen signals in the cytoplasm of A549 cells, which overlapped with MitoTracker staining ( Figure 3D). We previously found that blocking of the DDR resulted in the disappearance of dsDNA in the cytosol of Yac-1 cells (Lam et al, 2014). Similarly, inhibition of the DDR-initiating kinases ATM and ATR in A549 cells abrogated the cytosolic dsDNA staining ( Figure 3E).…”

“…Constitutive Presence of DNA in the Cytosol of Several Human Cell Lines We previously found that ssDNA and dsDNA are constitutively present in the cytosol of the mouse lymphoma cell line Yac-1 (Lam et al, 2014) and Em-Myc B cell lymphomas (Figures 1C and S2A). To extend these observations to human cell lines, we stained the human lung carcinoma cell line A549, the acute monocytic leukemia cell line THP1, and the lung fibroblast cell line MRC5 for the presence of cytosolic ssDNA and dsDNA (Figures 1D,S2B,and S2C).…”

“…DNA damage also activates IRF3 and induces the secretion of type I IFNs (Brzostek-Racine et al, 2011;Lam et al, 2014). We previously found that DNA is present in the cytosol of the mouse B-cell lymphoma cell line BC2, a B cell lymphoma cell line derived from Em-Myc mice, after DMSO (upper row) or 10 mM Ara-C (lower row) for 16 hr and co-stained for the mitochondrial marker COX IV (green) and ssDNA (left) or dsDNA (right) (red) in the presence of DAPI (blue).…”

“…Data are representative of more than three independent stainings. treatment with the genotoxic replication inhibitor cytosine b-D-arabinofuranoside hydrochloride (Ara-C) ( Figure 1A; Lam et al, 2014).…”

“…Type I IFNs induce the transcription of many anti-viral genes and activate important components of the innate and adaptive immune system, including natural killer (NK) cells, T cells, and B cells. NF-kB and IRF3 also are activated in response to DNA damage, and activation of the DDR induces the expression of type I IFNs (Brzostek-Racine et al, 2011;Lam et al, 2014). However, the mechanisms leading to the expression of type I IFNs in response to DNA damage have not been well characterized.…”